The need to separate small particles such as macromolecules, virus-like particles, bacteria, or colloids has become an important task preparatory to their characterization in terms of their mass or size and the determinations of the distributions thereof. In recent years, one of the most successful means by which such particles may be separated is by asymmetric flow field flow fractionation, AsFFFF, or A4F for short. The apparatus is a variant of the earlier cross flow FFF device described by J. Calvin Giddings in his 1993 Science paper, volume 260, pages 1456-1465. The A4F variation is described by its inventor Carl-Gustav Wahlund in his 1987 paper with J. Calvin Giddings “Properties of an asymmetrical flow field-flow fractionation channel having one permeable wall,” Analytical Chemistry 59, 1332-39.
An A4F unit is comprised of the following elements together with means to hold them together: 1) a bottom assembly structure holding a liquid-permeable frit, usually made from sintered stainless steel particles, 2) a permeable membrane that lies over the frit, 3) a spacer of thickness from about 75 to 800 μm containing a cavity, and 4) a top assembly structure generally holding a transparent plate of material such as Lexan® or glass. The resulting sandwich is held together with bolts or other means. The coffin-shaped cavity in the spacer will serve as the channel in which separation will occur. The top assembly structure contains three holes that pass through the generally transparent plate, called ports, that are centered above the channel and permit the attachments of fittings thereto. These ports are: 1) a mobile phase inlet port located near the beginning of the channel and through which is pumped the carrier liquid, the so-called mobile phase, 2) a sample port, very close to and downstream of the inlet port, into which an aliquot of the sample to be separated is introduced to the channel, and 3) an exit port through which the fractionated aliquot leaves the channel.
A4F channels are used to separate particles of the varying classes listed above and spanning a size range from a few nanometers to tens of micrometers. The separation of a sample aliquot comprised of such particles depends in turn on the length, breadth, and thickness of the coffin-shaped cavity. In addition, it depends on the channel flow rate, the ratio of the cross flow to channel flow, temperature, liquid viscosity, pH, ionicity, the physical composition of the particles themselves, and the type of permeable membrane lying over the frit. By suitably programming the time variation of the channel-to-cross flow ratio, separations of different particle classes may be improved significantly and often a great range of particle sizes present in the injected sample aliquot may be separated in the same run. Indeed, for each class of particles to be separated an optimal separation may be developed by empirically varying those variables accessible. The only variable that cannot be changed for a conventional channel is the channel length.
Historically, the channel length for A4F has been of the order of 25 to 30 cm with a greatest breadth of the order on 1 to 3 cm that tapers along its length and ends at a breadth comparable to the breadth of the exit port. Recent studies have suggested that a channel of shorter length would provide certain benefits and, on this basis, a completely new structure was developed and incorporated into a shorter A4F unit. However, with the choice of a single channel, the possibility of providing better separations for certain classes of particles can never be thoroughly studied unless all the affecting variables listed above may be tested. Thus effects of flow rates on the separation of each class of particle are easily tested using a fixed channel, but current channel design tradition does not allow for varying the length. It does permit variation of channel breadth and membrane thickness with the same three port locations. If only a single channel length is available, several questions arise: Is there a length that will produce better separations than that produced by either of the fixed length devices available? Is there an optimal length that will produce equivalent separations but require smaller sample aliquot size? May one combine the results obtained from several different lengths for the same samples to yield better characterizations of the samples examined? Does optimal separation of each sample type have an associated best channel length?
It is a basic objective of this invention to provide an apparatus and method by which these questions may be answered. Separation depends on many parameters, some of which are controlled by the software and the system operator. Until this invention, however, one parameter, the channel length, could not be varied. Thus it is a further object of the present invention to provide greater separation flexibility by which samples may be characterized more extensively by providing accessibility to different channel lengths within the same channel structure.